The TNF family proteins CD154 (CD40 Ligand), CD178 (Fas Ligand), and TNF related apoptosis inducing ligand (TRAIL) are known to play important roles in the modulation of inflammation and adaptive immune responses. Recent studies showed activated platelets express CD154, CD178, and TRAIL. Platelets, traditionally known for their haemostatic function, are known to play a key role in the initiation and modulation of the innate and inflammatory immune responses through the release of soluble and membrane bound factors at the site of vascular injury. With the discovery that platelets express potent immunomodulatory molecules such as CD154 and CD178, a role for platelets as a link between innate, inflammatory and adaptive immune responses is emerging. Despite the identification of immune regulatory functions for platelet-derived TNF proteins, there have no studies to date, investigating how platelets acquire and express TNF family proteins. The objective of the proposed study is to establish a foundation for understanding the mechanisms of TNF family proteins regulation in platelets and megakaryocytes. Our preliminary data provide evidence that TNF proteins are expressed by megakaryocytes and potentially regulated by the megakaryocyte-specific, GATA and the Ets families of transcriptional regulators. The specific aims outline a series of experiments designed to elucidate the regulatory pathways leading to TNF family protein expression by platelets. The first specific aim will address the hypothesis that GATA-1 and Fli-1 directly mediate CD154 expression in megakaryocytes. The second specific aim will address the hypothesis that CD178 (and TRAIL) is either directly regulated by GATA-1 or indirectly regulated by Ets factors. Further, the mechanism of how platelets acquire soluble CD178 will be addressed. Both specific aims will utilize well-established molecular approaches to achieve the objective of proposed research. Understanding the regulatory mechanisms by which the TNF family members are expressed in platelets may provide novel approaches for the development of therapies for chronic inflammatory diseases including atherosclerosis, inflammatory bowel disease, and interstitial cystitis.